POLE c.6634A>G, p.(Met2212Val)

Reasoning

PM2 at supporting strength requires the variant to be absent from, or at a frequency at or below 0.0001 in, a large control/general population cohort (supplied generic calibration; ACMG/AMP 2015, PMID:25741868, as refined by the ClinGen SVI PM2-supporting recommendation). This variant sits an order of magnitude below that threshold: gnomAD v4.1 all-comers total AF 1.23936e-06 (2/1,613,730 alleles), homozygotes 0, grpmax FAF 2.8e-07, highest observed ancestry European (non-Finnish) AF 1.69488e-06 (2/1,180,028 alleles). It is additionally absent from gnomAD v2.1, from the gnomAD v2.1 non-cancer exome and gnomAD v3.1 non-cancer genome subsets, and from gnomAD-Canada v1.0. PM2 is therefore met at supporting strength. The governing POLE framework specifies no gene-specific PM2 threshold and no non-cancer/exome-only population source, so the generic 0.0001 threshold and the all-comers gnomAD v2.1/v4.1 entries are the default; the verdict is unchanged under the non-cancer subsets (1.23936e-06 well below 0.0001 either way).

Rule

PM2 (supporting pathogenic): absent from, or at an allele/filtering frequency <= 0.0001 in, a large control/general population cohort (ACMG/AMP 2015 with ClinGen SVI supporting-strength calibration, PMID:25741868).

Evidence considered

• PM2 threshold applied from the supplied published calibration: supporting pathogenic strength when the variant is absent or at an allele/filtering frequency <= 0.0001 in a large control population (ACMG/AMP 2015, PMID:25741868; ClinGen SVI recommendation to downgrade PM2 to supporting).
• gnomAD v4.1 (all-comers totals; default source): variant chr12-132625668-T-C, total AF 1.23936e-06 (AC 2 / AN 1,613,730), homozygotes 0; grpmax FAF 2.8e-07; highest ancestry European (non-Finnish) AF 1.69488e-06 (AC 2 / AN 1,180,028), homozygotes 0; exome AF 1.3686e-06 (2/1,461,430), genome AF 0 (0/148,300).
• gnomAD v2.1 (all-comers totals; default source): variant 12-133202254-T-C, reported absent from gnomAD v2.1.
• gnomAD v2.1 non-cancer exomes (sensitivity analysis; reported by its exome-only figures): reported absent from gnomAD v2.1 non-cancer, corroborating the rarity.
• gnomAD v3.1 non-cancer genomes (sensitivity analysis; genomes only, v3 has no exome cohort): reported absent from gnomAD v3.1 non-cancer.
• gnomAD-Canada v1.0 (HostSeq genomes, small local dataset): query completed and reported this variant absent, treated as low-weight corroboration of rarity rather than as a primary frequency source.
• Source selection: the governing POLE framework is the local Leon-Castillo-derived custom framework (official_clingen = false), which specifies no non-cancer or exome-only population source for PM2, so the all-comers gnomAD v2.1/v4.1 entries are the default; the gnomAD v2.1 non-cancer exome and gnomAD v3.1 non-cancer genome subsets were nevertheless consulted and agree (no observed alleles).
• The variant is far below, not merely at the edge of, the 0.0001 rarity threshold: the highest single value anywhere (European non-Finnish AF 1.69488e-06) is ~59-fold below 0.0001, and the grpmax FAF (2.8e-07) is ~357-fold below.
• The single-submitter ClinVar classification (Uncertain significance, SCV002263622) was not used as evidence for PM2.

Sources

Reasoning

PP2 applies to a missense variant in a gene where missense variation is a common mechanism of pathogenicity and where the rate of benign missense variation is low. POLE satisfies the mechanism limb: the disease-causing POLE variants characterised in the governing source are missense substitutions in the exonuclease/proofreading domain (the five established hotspots P286R, V411L, S297F, A456P, S459F are all missense), and gene context notes germline POLE mutations causing polyposis/colorectal cancer predisposition are predominantly proofreading-domain missense changes. Because missense, not truncation, is the established pathogenic mechanism, the criterion is met at its default Supporting weight. The caveat is that this particular substitution (M2212V) lies outside the exonuclease domain, so the gene-level mechanism does not by itself carry domain-specific weight; a formal gene-level missense-constraint metric was not available in the case materials, which is why confidence is moderate rather than high.

Rule

PP2: missense variant in a gene with a low rate of benign missense variation where missense is a common mechanism of pathogenicity.

Evidence considered

• León-Castillo et al. 2020 (J Pathol 250:323-335): all five pathogenic, recurrent 'hotspot' POLE exonuclease-domain mutations (P286R, V411L, S297F, A456P, S459F) are missense substitutions, establishing missense as the pathogenic mechanism in POLE.
• Supplementary Table S2 (PATH-250-323-s003) tabulates missense POLE variants by in silico tool class, consistent with missense being the relevant variant class for POLE pathogenicity review.
• Gene context: POLE encodes the catalytic subunit of DNA polymerase epsilon; germline mutations cause polyposis and colorectal cancer predisposition, and the characterised disease alleles are proofreading-domain missense changes.

Still missing

• No formal gene-level low-rate-of-benign-missense-variation metric (e.g. gnomAD missense constraint / ClinGen PP2 gene list) was present in the case materials; the mechanism limb was supported directly while the frequency limb was inferred from the gene's established missense-driven pathogenicity.

Sources

Reasoning

Scope: NM_006231.4:c.6634A>G is a missense substitution (NP_006222.2:p.(Met2212Val)), so BP4 is assessed on the missense protein-impact path - REVEL only. The local REVEL v1.3 score is 0.212, which satisfies the ClinGen SVI BP4 supporting band (<=0.29) but is above the moderate (<=0.183) and strong (<=0.016) bands; BP4 is therefore met at Supporting strength only. This is not a gray-zone result: the indeterminate zone lies between the BP4 and PP3 cut-offs (>0.29 and <0.644), and 0.212 is outside it on the benign side. The POLE custom framework's BP4 rule (apply BP4_Supporting only when the exact missense variant appears in Supplementary Table S2 or S3, the REVEL class is 'Likely benign'/'Likely-benign', and the number of benign in silico results is >=4) cannot be applied because p.M2212V is absent from both tables, so the generic REVEL workflow governs as the framework itself directs. The SpliceAI result (max delta 0.014) was not used for BP4, since 'no predicted splice impact' is not BP4 support for a missense variant.

Rule

Generic REVEL BP4 supporting (ClinGen SVI, Pejaver et al. 2022, PMID:36413997); POLE custom BP4 rule does not apply (variant absent from Supplementary Tables S2/S3).

Evidence considered

• REVEL v1.3 local lookup (source_registry key 'revel') returned score 0.212 for chr12:132625668 T>C (NM_006231.4:c.6634A>G); this is the only predictor used for BP4, per the missense-path rule.
• ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997): BP4 supporting <=0.29, moderate <=0.183, strong <=0.016. 0.212 is <=0.29 but >0.183, so BP4 is met at Supporting strength.
• The labeled gray/indeterminate REVEL zone (>0.29 and <0.644) does not contain 0.212, so BP4 is not blocked by a gray-zone score.
• POLE custom framework BP4 rule requires the exact variant to be present in Supplementary Table S2 or S3 with REVEL class 'Likely benign'/'Likely-benign' and >=4 benign in-silico results; searches of PATH-250-323-s003.xlsx.txt and PATH-250-323-s004.xlsx.txt returned no entry for any notation of this variant, so the custom rule is unavailable and its own fallback to the generic workflow applies.
• BayesDel score -0.035 was available (source_registry key 'bayesdel') but was not used: no generic ACMG-strength calibration for BayesDel is established, and the governing POLE materials specify no BayesDel cut-off, so it is not_available for PP3/BP4.
• SpliceAI max delta 0.014 was not used for BP4; for a missense variant the splice path is not applicable.

Still missing

• The queried variant (c.6634A>G / p.Met2212Val / p.M2212V) has no entry in the VCEP in silico tables PATH-250-323-s003.xlsx (Supplementary Table S2) or PATH-250-323-s004.xlsx (Supplementary Table S3); both files were searched in full and contain no row for this substitution, so the gene-specific BP4 lookup (which also requires a count of >=4 benign in-silico tool results) could not be applied and the generic REVEL workflow was used instead.
• No POLE-specific REVEL/BP4 numeric cut-off exists in the governing materials; the generic ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997) was applied.

Sources

Reasoning

PS1 requires the same amino-acid change (p.Met2212Val) to have been previously established as pathogenic. This exact substitution is not present in any governing VCEP source: it is absent from the Leon-Castillo et al. 2020 PDF text and from VCEP Supplementary Tables S1 (PATH-250-323-s002), S2 (PATH-250-323-s003) and S3 (PATH-250-323-s004) after direct full-text searches for c.6634A>G, p.Met2212Val and p.M2212V. ClinVar (variation 1478557) records the variant only once, as Uncertain significance (single submitter, Labcorp/Invitae), i.e. not an established pathogenic assertion. No same-change pathogenic comparator therefore exists, so PS1 is not met.

Rule

PS1: same amino-acid change as a previously established pathogenic variant.

Evidence considered

• Searched the governing VCEP files for the exact same change (p.Met2212Val / c.6634A>G / p.M2212V): no entry in the Leon-Castillo 2020 paper text or in Supplementary Tables S1, S2 or S3.
• ClinVar variation 1478557 (NM_006231.4(POLE):c.6634A>G, p.Met2212Val) is classified Uncertain significance, criteria provided single submitter, 1 submission (SCV002263622, Labcorp Genetics), review status 1 star, last evaluated 2024-04-08.
• No previously established pathogenic occurrence of the identical p.Met2212Val substitution was found in any consulted source.

Sources

Reasoning

The governing specification for this variant is the León-Castillo et al. 2020 custom POLE framework, which outranks generic ACMG here. That framework adapts PS4 as a low-weight somatic-recurrence signal capped at Supporting and states the rule exactly: apply PS4_Supporting only when the exact POLE missense variant is (i) recurrent in BOTH the COSMIC and TCGA endometrial carcinoma cohorts in Supplementary Table S1, (ii) has a combined EC count >=10, and (iii) belongs to the paper's established/pathogenic set (p.P286R, p.V411L, p.A456P, p.S297F). The stated operator is a '>=' on the combined EC count and a membership requirement on the exact variant. I searched PATH-250-323-s002.xlsx (Supplementary Table S1) for all three notations (c.6634A>G, p.Met2212Val, p.M2212V) and found no row for this variant; the nearest listed entry is p.Q2217* (c.6649C>T, 1 EC in COSMIC), which is a different substitution at a different codon. The variant is likewise absent from the PATH-250-323.pdf body text. Independent recurrence checks agree: COSMIC (queried 'POLE c.6634A>G') returned no somatic record, OncoKB reports 'Unknown Oncogenic Effect' with no variant-specific evidence, and cancerhotspots.org found no significant hotspot at M2212. With no combined EC count to compare, the inequality cannot be satisfied: combined EC count (0/not listed) >= 10? no. Moreover c.6634A>G (p.Met2212Val) lies outside the POLE exonuclease domain (approx. residues 268-471), which the paper itself reports as generally non-pathogenic (only 1/23 non-exonuclease-domain tumours showed the ultramutated signature). No germline case-control enrichment evidence exists either, so the generic PS4 (prevalence in affecteds significantly increased vs controls) is also unmet. PS4 is not met.

Rule

León-Castillo 2020 custom POLE framework, PS4_Supporting: exact variant recurrent in BOTH COSMIC and TCGA EC cohorts in Supplementary Table S1, combined EC count >=10, and part of the paper's established pathogenic set {P286R, V411L, A456P, S297F}; capped at Supporting.

Evidence considered

• León-Castillo 2020 custom POLE framework PS4_Supporting rule (quoted operator '>=' applied to combined EC count; membership requirement on the exact variant): exact variant must recur in both COSMIC and TCGA EC cohorts in Supplementary Table S1 with combined EC count >=10 and belong to {P286R, V411L, A456P, S297F}; capped at Supporting.
• PATH-250-323-s002.xlsx (Supplementary Table S1) searched for c.6634A>G, p.Met2212Val and p.M2212V: no row. Nearest entries: p.P286R (181 ECs), p.V411L (114), p.A456P (20), p.S297F (18), p.P436R (5), p.S459F (4), p.A465V (4); the only nearby residue, p.Q2217* (c.6649C>T, 1 EC), is a different variant.
• PATH-250-323.pdf full text searched for c.6634A>G, p.Met2212Val and p.M2212V: absent. The paper reports only 1/23 (4%) tumours with non-exonuclease-domain mutations showed the ultramutated signature, concluding most mutations outside the exonuclease domain are not pathogenic - relevant context because M2212 lies outside the exonuclease domain.
• COSMIC query 'POLE c.6634A>G' returned no result ('This variant has not previously been reported in somatic cancers'); cancerhotspots.org found no statistically significant hotspot at M2212; OncoKB returned 'Unknown Oncogenic Effect' with no variant-specific reviewed evidence. No somatic recurrence is therefore available to substitute for the absent Table S1 row.
• No germline case-control enrichment statistic (odds ratio, affected-vs-control prevalence) exists for this variant in any source, so even the generic ACMG PS4 prevalence comparison cannot be evaluated as met.

Still missing

• Searched PATH-250-323-s002.xlsx (Supplementary Table S1) for c.6634A>G, p.Met2212Val and p.M2212V: no entry exists for this variant, so the custom PS4 recurrence rule cannot be triggered by pre-assigned tables.
• Searched PATH-250-323.pdf.txt (full text of León-Castillo et al. 2020) for c.6634A>G, p.Met2212Val and p.M2212V: no entry/mension of this variant.
• PATH-250-323-s001.tif (supplementary figure, declared for PS4) could not be converted to searchable text and was not visually inspected; it is a visual summary and does not add variant-specific recurrence counts beyond Table S1.
• No germline case-control cohort or affected-vs-control prevalence data for this variant were available in any case source (no COSMIC record; COSMIC has no source_registry key, so the negative COSMIC result is recorded here rather than in evidence_refs).

Sources

Reasoning

The variant is p.Met2212Val at codon 2212, outside POLE's exonuclease (proofreading) domain. The governing custom POLE framework derived from Leon-Castillo et al. 2020 restricts PM1 to specific exonuclease-domain residues: PM1_Strong for the five established hotspots p.P286R, p.V411L, p.S297F, p.A456P and p.S459F; PM1_Moderate for the exact non-hotspot exonuclease-domain variants p.F367S, p.L424I, p.M295R, p.P436R, p.M444K and p.D368Y; and PM1_Supporting for the exact recurrent exonuclease-domain variants p.A465V, p.L424V, p.T278M and p.A428T. M2212 is not one of these and is not in the exonuclease domain, which by the paper's own analysis spans roughly the N-terminal proofreading region (72% of POLE mutations studied mapped within it; the five hotspots are all exonuclease domain). Residue 2212 sits near the C-terminus of the 2286-residue protein, far outside the domain; the closest table entry, p.Q2217* (c.6649C>T), is a different, non-recurrent codon. vcep_materials.json's domain_tables key is empty ({}), so there is no additional authoritative approved-domain list that would capture this residue. The paper explicitly states that a large majority of mutations outside the exonuclease domain are not pathogenic, arguing against a hotspot/domain call here. PM1 is therefore not met.

Rule

PM1: missense variant in a mutational hotspot and/or critical functional domain without benign variation (governed by the POLE custom framework residue-specific PM1 rules).

Evidence considered

• León-Castillo et al. 2020 (J Pathol 250:323-335) designates five exonuclease-domain hotspot missense mutations as pathogenic: P286R (21 cases), V411L (13), S297F (3), A456P (2), S459F (2), from 59 exonuclease-domain mutations among 82 somatic POLE-mutant endometrial cancers.
• The same paper states a large majority of mutations outside the exonuclease domain are not pathogenic and behave as passengers; residue 2212 is outside the exonuclease domain.
• The governing custom POLE framework customizes PM1 with explicit residue lists (hotspot Strong set; non-hotspot Moderate set F367S/L424I/M295R/P436R/M444K/D368Y; Supporting set A465V/L424V/T278M/A428T); p.M2212V is not on any list.
• Searched Supplementary Table S1 (PATH-250-323-s002) for the residue: no M2212 entry; nearest row is p.Q2217*/c.6649C>T, a different codon.
• CancerHotspots query POLE M2212 returned no statistically significant hotspot row.

Still missing

• vcep_materials.json domain_tables is empty ({}), so no governed critical-domain coordinate list was available; PM1 was assessed against the Leon-Castillo 2020 custom framework's residue-specific rules and the paper's exonuclease-domain/non-domain distinction instead.
• PATH-250-323-s001.tif (declared for PM1) could not be converted to text and was not visually reviewed.

Sources

Reasoning

PM5 requires a different missense change at the same residue (M2212) to be a previously established pathogenic variant. No such comparator exists. ClinVar reports only the queried change itself (p.Met2212Val, Uncertain significance), with no other pathogenic M2212 substitution. The automatically harvested comparator set (pm5_candidates.json) returned 0 candidates and explicitly could not confirm same-residue PM5 semantics. The governing VCEP tables contain no M2212 variant at all. Searches of the paper and Tables S1-S3 for p.M2212V/p.Met2212Val/c.6634A>G returned nothing; the only nearby table entry is p.Q2217* at codon 2217, which is a distinct residue and a nonsense change. PM5 is not met.

Rule

PM5: missense change at the same amino-acid residue as a previously established pathogenic variant, but a different amino-acid change.

Evidence considered

• ClinVar variation 1478557 contains a single submission for p.Met2212Val classified Uncertain significance; no other variant at codon 2212 is listed.
• pm5_candidates.json: eligible_for_classic_pm5_search false, total_candidates_seen 0, same_residue_candidates 0, recommendation 'not_applicable' - no same-residue comparator identified.
• Searched VCEP Supplementary Tables S1, S2 and S3 for p.M2212V/p.Met2212Val/c.6634A>G: no same-residue entry; nearest is p.Q2217* (c.6649C>T).

Still missing

• No same-residue (M2212) comparator missense variant of any classification was found in ClinVar or in the governing VCEP supplementary tables; the closest residue-level datum is p.Q2217* (c.6649C>T), a different codon.

Sources

Reasoning

Scope: NM_006231.4:c.6634A>G is a missense substitution (NP_006222.2:p.(Met2212Val), consequence_class = missense), so the applicable computational path is the missense protein-impact path - REVEL only. The local REVEL v1.3 lookup returned 0.212, which is below the ClinGen SVI supporting PP3 threshold of >=0.644 (Pejaver et al. 2022, PMID:36413997); it also falls short of the moderate (>=0.773) and strong (>=0.932) bands, so PP3 is not met at any strength. The governing POLE custom framework's PP3 rule (apply PP3_Supporting only when the exact missense variant appears in Leon-Castillo Supplementary Table S2 or S3, the REVEL class is 'likely disease causing', and the number of benign in silico results is <=1) does not apply: p.M2212V is absent from both Supplementary Table S2 (TCGA EC) and Supplementary Table S3 (non-TCGA EC cohorts), and the framework itself instructs falling back to the generic in-silico workflow in that case. SpliceAI was not consulted for this criterion because this is a missense variant, for which splice prediction carries no calibrated weight; its max delta (0.014) was neither used nor needed.

Rule

Generic REVEL PP3 (ClinGen SVI, Pejaver et al. 2022, PMID:36413997); POLE custom PP3 rule does not apply (variant absent from Supplementary Tables S2/S3).

Evidence considered

• REVEL v1.3 local lookup (source_registry key 'revel') returned score 0.212 for chr12:132625668 T>C (NM_006231.4:c.6634A>G), found = true; this is the single missense-path predictor used for PP3.
• ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997): PP3 supporting >=0.644, moderate >=0.773, strong >=0.932. REVEL 0.212 is below all three, so PP3 is not met.
• POLE custom framework PP3 rule: PP3_Supporting is conditioned on the exact missense variant appearing in Supplementary Table S2 or S3 with REVEL class 'likely disease causing' (including spelling variants) and <=1 benign in silico result; the rule explicitly instructs falling back to the generic in-silico workflow if the variant is absent.
• Searched PATH-250-323-s003.xlsx.txt (Table S2, 44 TCGA variants) and PATH-250-323-s004.xlsx.txt (Table S3, 38 non-TCGA variants) for every notation of this variant (c.6634A>G, p.Met2212Val, p.M2212V, and by residue number 2212): no row matches. The codon-2212 neighbours present in the tables are unrelated substitutions (e.g. p.Q2217* in Table S3).
• Searched PATH-250-323.pdf.txt for the same notations; no occurrence of the variant and no gene-specific PP3 threshold table.
• SpliceAI max delta (0.014) was deliberately not used, because PP3/BP4 for a missense variant must come from REVEL only and a splice prediction cannot support a missense PP3.

Still missing

• The queried variant (c.6634A>G / p.Met2212Val / p.M2212V) has no entry in the VCEP in silico tables PATH-250-323-s003.xlsx (Supplementary Table S2) or PATH-250-323-s004.xlsx (Supplementary Table S3); both files were searched in full and contain no row for this substitution, so the gene-specific PP3 lookup could not be applied and the generic REVEL workflow was used instead.
• PATH-250-323.pdf (declared for PP3) contains no entry for c.6634A>G / p.Met2212Val / p.M2212V and no POLE-specific computational rule specification; it is contextual hotspot/interpretation material only.

Sources

Reasoning

PP5 requires a reputable independent source reporting the exact variant as pathogenic, and per the applied rule for this group only an exact-variant ClinVar expert-panel classification of Pathogenic or Likely pathogenic may trigger it; ordinary laboratory submissions, aggregate ClinVar labels and non-expert assertions must never trigger it. The exact variant has one ClinVar record (VariationID 1478557, NM_006231.4:c.6634A>G, p.Met2212Val). Its classification is 'Uncertain significance', review status 'criteria provided, single submitter', review stars 1, with a single submitter (Labcorp Genetics, formerly Invitae), origin germline, no expert-panel submission (expert_panel_submissions = 0), conditions 'not provided'. A single-submitter VUS is neither an expert-panel assertion nor a pathogenic assertion, so it cannot support PP5 at any strength. The only PMID cited by that submission is 28492532 (Nykamp et al. 2017, 'Sherloc'), a methodology/guidelines paper that does not mention this variant (variant_mentioned = false) and yields no variant-level classification. No other reputable source (OncoKB: Unknown Oncogenic Effect; COSMIC: no record) reports this variant as pathogenic. PP5 is not met.

Rule

PP5: reputable source reports the exact variant as pathogenic; per group rule restricted to exact-variant ClinVar expert-panel Pathogenic/Likely pathogenic classifications only (non-expert/aggregate submissions excluded).

Evidence considered

• ClinVar record for the exact variant: VariationID 1478557, NM_006231.4(POLE):c.6634A>G (p.Met2212Val), classification 'Uncertain significance', review status 'criteria provided, single submitter', 1 review star, 1 submission (SCV002263622, Labcorp Genetics/formerly Invitae, germline), expert_panel_submissions = 0, condition 'not provided'.
• The ClinVar submission cites only PMID 28492532 (Nykamp et al. 2017, Sherloc), a methodology refinement of the ACMG/AMP criteria that does not mention this variant and provides no variant-level classification.
• OncoKB returned 'Unknown Oncogenic Effect' with no variant-specific reviewed evidence, and COSMIC returned no record for this variant; neither source reports the variant as pathogenic.
• Applied rule for this group: PP5 triggers only on an exact-variant ClinVar expert-panel Pathogenic/Likely pathogenic classification; ordinary single-laboratory submissions must not trigger it.

Still missing

• No ClinVar expert-panel (3-star) classification exists for this variant, which is the only qualifying trigger for PP5 under the applied rule; if a VCEP/EP review is published in future, PP5 should be re-evaluated.

Sources

Reasoning

POLE NM_006231.4:c.6634A>G (NC_000012.11:g.133202254T>C; GRCh38 chr12:132625668 T>C; p.Met2212Val) is exceedingly rare in every population dataset examined. gnomAD v4.1 all-comers: total AF 1.23936e-06 (2/1,613,730 alleles, 0 homozygotes), grpmax FAF 2.8e-07, highest observed ancestry European (non-Finnish) AF 1.69488e-06 (2/1,180,028 alleles). gnomAD v2.1: absent. The BA1 stand-alone benign threshold requires an allele frequency >= 0.05 in a control/general population dataset (ACMG/AMP 2015, PMID:25741868). The single highest frequency observed anywhere in this case is 1.69488e-06, roughly 30,000-fold below 0.05, and no dataset or ancestry reaches even 0.001. Absence from the gnomAD v2.1 non-cancer exome and gnomAD v3.1 non-cancer genome subsets, and from gnomAD-Canada v1.0, is consistent with this. BA1 is therefore not met, and the verdict is identical whether the all-comers gnomAD v2.1/v4.1 totals or the non-cancer subsets are used. The ClinVar record for this variant was not used as evidence for this criterion.

Rule

BA1 (stand-alone benign): allele frequency >= 0.05 in a control/general population dataset (ACMG/AMP 2015, PMID:25741868).

Evidence considered

• gnomAD v4.1 (all-comers totals; default source because the governing POLE framework specifies no population source): variant chr12-132625668-T-C, total AF 1.23936e-06 (AC 2 / AN 1,613,730), homozygotes 0; grpmax FAF 2.8e-07; highest ancestry European (non-Finnish) AF 1.69488e-06 (AC 2 / AN 1,180,028), homozygotes 0; exome AF 1.3686e-06 (2/1,461,430), genome AF 0.
• gnomAD v2.1 (all-comers totals; default source): variant 12-133202254-T-C, reported absent from gnomAD v2.1 (no allele count, no homozygotes).
• gnomAD v2.1 non-cancer exomes (sensitivity analysis): reported absent from gnomAD v2.1 non-cancer; no allele count or frequency is available, so it contributes no signal above the all-comers result.
• gnomAD v3.1 non-cancer genomes (sensitivity analysis; v3 has no exome cohort, so this source is genomes-only): reported absent from gnomAD v3.1 non-cancer, with no allele count or frequency available.
• gnomAD-Canada v1.0 (HostSeq genomes, small local dataset): reported absent; treated as low-weight corroboration only, not as a frequency-based negative for BA1.
• BA1 threshold applied from the supplied published calibration: stand-alone benign at allele frequency >= 0.05 in a control/general population dataset (ACMG/AMP 2015, PMID:25741868). Threshold comparison used the highest ancestry-specific frequency (1.69488e-06) and the grpmax FAF (2.8e-07); both are ~4-5 orders of magnitude below 0.05, so BA1 fails decisively in every source.
• Ancestry pattern is not a single-population founder signal: the variant is observed only in European (non-Finnish) samples in gnomAD v4.1 (2/1,180,028), is below or absent in every other ancestry group, and has 0 homozygotes everywhere; the rarity is a variant-level property, not an artefact of one subpopulation.
• Governing framework check: no ClinGen CSPEC exists for POLE (cspec found = false), and the local POLE custom framework derived from Leon-Castillo et al. 2020 (official_clingen = false) customizes only PM1, PS4, PP3 and BP4, defining no BA1 threshold and no population source, so the generic ACMG/AMP 0.05 threshold and the all-comers gnomAD totals govern.
• A single-submitter ClinVar classification (Uncertain significance, SCV002263622) exists for this variant but was not used as evidence for BA1: a criterion is never inferred from another source's final classification.

Sources

Reasoning

BS1 (strong benign) requires an allele frequency greater than that expected for the disorder. No gene-specific population threshold exists for POLE: there is no ClinGen CSPEC for this gene, and the governing local POLE custom framework (leon-castillo-2020-custom-framework-v1, official_clingen = false) customizes only PM1, PS4, PP3 and BP4 and defines no BA1/BS1/BS2/PM2 thresholds, expressly falling back to generic ACMG/AMP outside its rules. The applicable threshold is therefore the generic SVI default of >= 0.01 (per-gene Whiffin-style derivation preferred, PMID:28518168). Against that threshold the variant falls far short: the highest frequency observed anywhere is 1.69488e-06 (gnomAD v4.1 all-comers, European non-Finnish, 2/1,180,028), with grpmax FAF 2.8e-07; gnomAD v2.1 is absent, and the gnomAD v2.1 non-cancer exome and gnomAD v3.1 non-cancer genome subsets are likewise absent. Every available value is roughly 6,000-fold or more below 0.01 and none reaches even 0.001, so BS1 is not met under the applicable threshold, and the same conclusion holds whether all-comers totals or non-cancer subsets are used. A caveat is that a per-gene derived threshold could in principle sit below the generic default and was not derivable from the materials available.

Rule

BS1 (strong benign): allele frequency greater than expected for the disorder; generic SVI default >= 0.01, per-gene Whiffin-style derivation preferred but not derivable here (PMID:28518168).

Evidence considered

• BS1 threshold applied from the supplied published calibration: strong benign at allele frequency >= 0.01, generic SVI default with per-gene derivation preferred (Whiffin et al. 2017, PMID:28518168).
• Frequency values used for the comparison, all far below 0.01: gnomAD v4.1 all-comers total AF 1.23936e-06 (2/1,613,730) and grpmax FAF 2.8e-07; gnomAD v2.1 all-comers absent; gnomAD v2.1 non-cancer exomes absent; gnomAD v3.1 non-cancer genomes absent.
• Highest ancestry-specific frequency in any dataset: European (non-Finnish) 1.69488e-06 (2/1,180,028) in gnomAD v4.1 all-comers. Even this maximum is ~6,000-fold below the 0.01 generic BS1 threshold and ~30,000-fold below 0.05.
• No homozygotes are reported in any dataset (gnomAD v4.1 total_hom 0; European non-Finnish hom 0; gnomAD v2.1 absent; non-cancer subsets absent), so the frequency signal rests entirely on two heterozygote carrier alleles.
• Governing framework check: no ClinGen CSPEC exists for POLE (cspec found = false), and the local POLE framework derived from Leon-Castillo et al. 2020 customizes only PM1, PS4, PP3 and BP4, stating that outside its explicit rules the interpreter should fall back to generic ACMG/AMP; no gene-specific population threshold or population source was available to override the generic default.
• Gene-level disease context (adult-onset, incompletely penetrant cancer-predisposition/polyposis phenotype) makes the 'greater than expected for the disorder' comparison inherently approximate for this gene; because no prevalence or penetrance figures were available in the case materials, the generic threshold was used and the derivation shortfall is recorded as a gap.

Still missing

• No per-gene (Whiffin-style) BS1 threshold could be derived for POLE: the governing local POLE framework specifies no population thresholds, and the case materials contain no POLE-associated disease prevalence, penetrance or allelic-heterogeneity inputs required for that derivation. The supplied generic SVI default (>= 0.01) was applied; the ~6,000-fold margin makes the 'not met' verdict insensitive to any plausible downward adjustment of the threshold.

Sources

Reasoning

BP1 applies only to a missense variant in a gene where truncating (loss-of-function) variants, and not missense variants, are the established disease mechanism. POLE is the opposite case: its pathogenic variants are missense substitutions in the exonuclease/proofreading domain (the five established hotspots P286R, V411L, S297F, A456P and S459F are all missense), and germline POLE disease alleles are similarly missense proofreading changes. Missense variation is therefore a recognised pathogenic mechanism for POLE, so the gene is not a truncating-only gene and BP1 is not met. This is the mutually exclusive counterpart to the PP2 mechanism assessment.

Rule

BP1: missense variant in a gene for which only truncating variants are an established mechanism of disease.

Evidence considered

• León-Castillo et al. 2020 (J Pathol 250:323-335): pathogenic POLE mutations are missense exonuclease-domain substitutions (P286R, V411L, S297F, A456P, S459F), demonstrating that missense, not truncation only, causes POLE-associated pathogenicity; thus the BP1 premise (truncating-only gene) fails.

Sources

Reasoning

BP6 requires a reputable independent source reporting the exact variant as benign, and per the applied rule for this group only an exact-variant ClinVar expert-panel classification of Benign or Likely benign may trigger it; ordinary laboratory submissions, aggregate ClinVar labels and non-expert assertions must never trigger it. The exact variant's single ClinVar record (VariationID 1478557, NM_006231.4:c.6634A>G, p.Met2212Val) is classified 'Uncertain significance' with review status 'criteria provided, single submitter', 1 review star, one submitter (Labcorp Genetics, formerly Invitae), origin germline, and expert_panel_submissions = 0. A single-submitter VUS is not a Benign/Likely benign assertion and is not expert-panel derived, so it cannot support BP6 at any strength. No other source asserts benignity for this variant (OncoKB: Unknown Oncogenic Effect; COSMIC: no record). The submission's only cited PMID (28492532, Sherloc methodology paper) does not mention the variant. BP6 is not met.

Rule

BP6: reputable source reports the exact variant as benign; per group rule restricted to exact-variant ClinVar expert-panel Benign/Likely benign classifications only (non-expert/aggregate submissions excluded).

Evidence considered

• ClinVar record for the exact variant: VariationID 1478557, NM_006231.4(POLE):c.6634A>G (p.Met2212Val), classification 'Uncertain significance', review status 'criteria provided, single submitter', 1 review star, single submitter (SCV002263622, Labcorp Genetics/formerly Invitae, germline), expert_panel_submissions = 0.
• The ClinVar submission cites only PMID 28492532 (Nykamp et al. 2017, Sherloc), a methodology paper that does not mention this variant and gives no variant-level benign classification.
• OncoKB returned 'Unknown Oncogenic Effect' and COSMIC returned no record for this variant; no source reports it as benign.
• Applied rule for this group: BP6 triggers only on an exact-variant ClinVar expert-panel Benign/Likely benign classification; ordinary single-laboratory submissions must not trigger it.

Still missing

• No ClinVar expert-panel (3-star) Benign/Likely benign classification exists for this variant, which is the only qualifying trigger for BP6 under the applied rule; re-evaluate if such a review is published.

Sources

Reasoning

PS2 (confirmed de novo) requires that the variant be demonstrated de novo in a patient with the disease phenotype and no family history, with parental testing showing that neither biological parent carries the variant; the same observation in a patient with a consistent family history is PS2 with a supporting-strength adjustment. No governing gene-specific specification exists for POLE: no ClinGen CSPEC/VCEP rule set was found, and the local custom POLE framework (Leon-Castillo et al. 2020) customizes only PM1, PS4, PP3 and BP4, so PS2 falls back to generic ACMG/AMP 2015 (Richards et al.). This case contains no proband, no pedigree, no family history, and no parental genotype results; nothing in the case documents a de novo occurrence or any transmission data. The criterion therefore cannot be evaluated and is recorded as not_assessed with the missing family-level data identified as the gap.

Rule

Generic ACMG/AMP 2015 (Richards et al., PMID:25741868) PS2 - confirmed de novo in a patient with the disease and no family history; no POLE gene-specific PS2 specification available.

Evidence considered

• Framework check: no ClinGen CSPEC/VCEP specification exists for POLE, and the local custom POLE framework (Leon-Castillo et al. 2020) customizes only PM1, PS4, PP3 and BP4; PS2 has no gene-specific specification, so generic ACMG/AMP 2015 (Richards et al., PMID:25741868) definitions apply.
• Case evidence (case_summary.json, evidence.json, prefetch.json, literature_pass.json) records no proband, no pedigree, no family history, and no parental genotype data for this variant.
• ClinVar variation 1478557 reports uncertain significance from a single submitter with no de novo or family-segregation information provided.

Still missing

• No proband is defined for this case; there is no affected individual linked to this variant.
• No parental testing (trio/mother-father genotypes) is reported, so a de novo occurrence cannot be confirmed or excluded.
• No family history or pedigree is documented to establish whether the occurrence is sporadic or consistent with an inherited pattern.

Sources

Reasoning

Not assessed. PS3 requires well-established functional studies demonstrating a damaging effect on the gene or gene product, with documented assay validity, reproducibility and biological relevance (generic ACMG/AMP 2015, Richards et al., PMID:25741868). No such study exists for POLE NM_006231.4:c.6634A>G (NP_006222.2:p.Met2212Val). No ClinGen CSPEC/VCEP specification exists for POLE (cspec.found = false); the governing specification is a local, non-official framework derived from Leon-Castillo et al. 2020 (framework_mode custom_gene_framework; official_clingen = false; precedence_rank 2) whose customized_criteria are exactly PM1, PS4, PP3 and BP4. It defines no PS3 rule and no POLE-specific PS3 strength calibration, so PS3 reverts to generic ACMG/AMP with no gene-specific weighting. The Leon-Castillo paper itself contains no laboratory functional assay: its own method for assessing the 'functional status' of POLE mutations is six in-silico tools (SIFT, PROVEAN, PolyPhen-2, PANTHER, SNAP2, REVEL), and computational prediction is by definition PP3/BP4 evidence, not the experimentally established functional evidence PS3 requires. p.Met2212Val is not mentioned anywhere in that paper (zero text matches for '2212' or '6634') and is absent from its Supplementary Tables S1-S3, which were read directly. OncoKB returns 'Unknown Oncogenic Effect' with is_variant_specific = false and no citations, i.e. an explicit record that no variant-specific reviewed functional evidence was identified. The only literature triaged for this case is PMID:28492532 (Sherloc), a criteria-refinement methods paper that does not mention this variant and reports no assay. Residue 2212 lies in the C-terminal polymerase region, outside the exonuclease domain and outside the five established exonuclease-domain hotspots (p.P286R, p.V411L, p.S297F, p.A456P, p.S459F), so no hotspot functional inference is available either. Because no qualifying functional study exists in any direction, PS3 is not assessed rather than not met; the absent evidence must not be read as a damaging-effect result.

Rule

Generic ACMG/AMP 2015 (Richards et al., PMID:25741868): PS3 = well-established functional studies show a damaging effect on gene/protein function. The governing local POLE framework (Leon-Castillo et al. 2020) specifies no PS3 rule and no official ClinGen POLE CSPEC/VCEP was available.

Evidence considered

• Governing specification: no ClinGen CSPEC exists for POLE (cspec.found = false), so the case uses a local, non-official POLE framework derived from Leon-Castillo et al. 2020 (framework_label 'Leon-Castillo et al. 2020 custom POLE framework'; framework_mode custom_gene_framework; official_clingen = false; precedence_rank 2). Its customized_criteria list is exactly [PM1, PS4, PP3, BP4]; no PS3 trigger or strength rule is defined, and the framework notes state that outside the explicit paper-derived rules it falls back to generic ACMG/AMP. PS3 is therefore evaluated under generic ACMG/AMP 2015 (Richards et al., PMID:25741868), which requires well-established functional studies showing a damaging effect.
• Variant: POLE NM_006231.4:c.6634A>G, NP_006222.2:p.(Met2212Val) (NC_000012.11:g.133202254T>C), a missense substitution at residue 2212 in the C-terminal polymerase region. It is outside the exonuclease domain and is not one of the five established exonuclease-domain hotspot substitutions (p.P286R, p.V411L, p.S297F, p.A456P, p.S459F), so no hotspot-based functional inference applies.
• OncoKB record for POLE M2212V (https://www.oncokb.org/gene/POLE/M2212V): classification 'Unknown Oncogenic Effect', is_variant_specific = false, not_reviewed = true, citations_pmids = []. OncoKB explicitly states it did not identify variant-specific reviewed functional evidence, so it supplies no damaging-effect assay claim for PS3.
• Leon-Castillo et al. 2020 full text (J Pathol 250:323-335; PATH-250-323.pdf) was read directly: it contains no laboratory functional assay for any variant, and its stated approach to variant 'functional status' is in-silico only - 'To evaluate the functional status of somatic POLE mutations, we used six widely-used in silico tools: SIFT, PROVEAN, PolyPhen-2, PANTHER, SNAP2, and the meta predictor REVEL.' The strings '2212' and '6634' occur 0 times in the full text, so p.Met2212Val is never named.
• Supplementary tables were opened directly: PATH-250-323-s002.xlsx (Table S1, 101 recurrent POLE variants with COSMIC/TCGA EC counts), PATH-250-323-s003.xlsx (Table S2, 44 TCGA in-silico rows) and PATH-250-323-s004.xlsx (Table S3, 38 non-hotspot in-silico rows). p.Met2212Val / c.6634A>G is absent from all three; the nearest S1 entry is the unrelated p.Q2217* (c.6649C>T). These tables hold somatic recurrence counts and computational predictions only, never a functional assay, so they cannot supply PS3 evidence.
• Literature: the only PMID triaged for this case is 28492532 (Sherloc, Nykamp et al. 2017), a criteria-refinement methods paper with no variant-level data and variant_mentioned = false. No full-text publication was fetched or extracted (paper_extracts.json and fulltext_map.json are empty), and no functional assay of p.Met2212Val was found anywhere in the corpus.
• Not used for PS3: the paper's non-exonuclease-domain observations ('Only 1/23 (4%) tumours with non-EDM showed these genomic alterations, indicating that a large majority of mutations outside the exonuclease domain are not pathogenic'; and that >95% of non-exonuclease-domain POLE mutations score as non-pathogenic by POLE-score) are somatic tumour genomic-signature findings in endometrial carcinoma, not a validated assay of p.Met2212Val, and cannot be scored as PS3.
• No numeric threshold applies: PS3 is a qualitative evidence-existence criterion in ACMG/AMP 2015 and no POLE-specific PS3 calibration is provided by the governing materials.

Still missing

• No laboratory functional assay of POLE p.Met2212Val (e.g. polymerase-epsilon proofreading or extension fidelity, yeast/cell-based mutator-complementation assay, or an equivalent validated readout) was identified in any retrieved source, so the PS3 damaging-effect requirement cannot be evaluated.
• The governing POLE framework (Leon-Castillo et al. 2020-derived, non-official local framework) defines no PS3 rule and provides no POLE-specific PS3 strength calibration; no official ClinGen POLE CSPEC/VCEP specification was available.
• No variant-level functional report for c.6634A>G could be traced through the ClinVar citation trail; the single cited PMID (28492532) is a methodology paper and does not mention the variant.

Sources

Reasoning

PM3 is a recessive-modality criterion: it requires that POLE NM_006231.4:c.6634A>G (p.Met2212Val), a missense change in exon 47, be shown to lie in trans with a pathogenic variant in the same individual. The governing specification for this case is a local, literature-derived POLE package (Leon-Castillo et al. 2020, somatic endometrial-carcinoma hotspot context) that customises only PM1, PS4, PP3 and BP4 and sets no PM3 rule, and no ClinGen CSPEC/VCEP exists for POLE (cspec found = false), so the generic ACMG/AMP 2015 definition applies (PMID:25741868). POLE has both dominant germline contexts (polymerase proofreading-associated polyposis / colorectal-cancer predisposition) and biallelic recessive ones (the POLE-deficiency / IMAGE-I syndrome), so a trans observation would be admissible if one existed; none is documented in any source available for this variant. The ClinVar record (variation 1478557, VCV001478557) contains a single germline submission (SCV002263622, Labcorp Genetics; Uncertain significance; review status 'criteria provided, single submitter'; no expert panel) whose condition is recorded only as 'not provided', and reports no second POLE variant, no zygosity, no phase and no affected relative. The population datasets report zero homozygotes (gnomAD v4.1 2/1,613,730 alleles, AF 1.24e-06, European non-Finnish 2/1,180,028, homozygotes 0; gnomAD v2.1 absent; the gnomAD-Canada v1.0 query returned no record), so no individual is reported in whom a second allele could be examined. No full text was available for the single PMID cited by ClinVar (PMID:28492532) and no paper extract exists for this case, so no case-, phase- or family-level data could be drawn from the literature. With no proband genotype, no candidate trans partner and no phasing data, the configuration PM3 requires is untested and the criterion is recorded as not assessed rather than met or refuted.

Rule

Generic ACMG/AMP PM3 (PMID:25741868): for recessive disorders, the variant is detected in trans with a pathogenic variant; applied generically because the governing local POLE package specifies only PM1, PS4, PP3 and BP4 and no ClinGen CSPEC/VCEP exists for POLE.

Evidence considered

• Governing framework: the local POLE package (Leon-Castillo et al. 2020 custom POLE framework, source 'local_custom_framework', official_clingen false, precedence_rank 2) customises only PM1, PS4, PP3 and BP4 for exonuclease-domain hotspot/recurrence and table-grounded in-silico evidence and states explicitly that outside those rules generic ACMG/AMP applies; it contains no PM3 rule. No ClinGen CSPEC/VCEP for POLE was retrieved (cspec found = false), so PM3 was evaluated under the generic ACMG/AMP 2015 definition: for recessive disorders, the variant detected in trans with a pathogenic variant (PMID:25741868).
• ClinVar variation 1478557 (VCV001478557) for NM_006231.4(POLE):c.6634A>G (p.Met2212Val): one germline submission from a clinical laboratory (SCV002263622, Labcorp Genetics, formerly Invitae), classification Uncertain significance, review status 'criteria provided, single submitter' (1 star), no expert-panel submission, condition recorded only as 'not provided', cited PMID 28492532. The submission provides no second POLE variant, no zygosity, no phase and no relative genotype. The submitted classification itself was not used to infer PM3, since a criterion may not be derived from another source's final classification.
• Inheritance context for the gene: the case's gene-level artifact (pvs1_gene_context) records germline_disease_context_found = true but carries an empty inheritance list and no inheritance-mode entry for POLE, and the ClinVar condition attached to this variant is 'not provided'. POLE has both dominant germline contexts (polymerase proofreading-associated polyposis / colorectal-cancer predisposition) and biallelic recessive ones (POLE-deficiency / IMAGE-I syndrome), so neither a dominant-only nor a recessive-only reading is fixed for this variant and the trans configuration PM3 asks about remains open.
• Population data: gnomAD v4.1 2/1,613,730 alleles (AF 1.23936e-06; highest subpopulation European non-Finnish 2/1,180,028, AF 1.69488e-06; grpmax FAF 2.8e-07; homozygotes 0); gnomAD v2.1 returned no record; gnomAD-Canada v1.0 returned no record. No dataset reports a homozygote, so there is no population-level observation of this allele on both chromosomes from which any allelic configuration could be inferred.
• Somatic data: the COSMIC search returned no record of POLE c.6634A>G, so no co-occurring somatic alteration from which a cis/trans signal might be read is available for this variant.
• Case-level literature: the single PMID cited by the ClinVar submission (PMID:28492532, the Sherloc refinement of the ACMG/AMP criteria) was not available as full text for this case and no paper extract exists, so it supplies no variant-level, phase, segregation or family data for c.6634A>G.
• The local POLE package underlying the framework (PATH-250-323 PDF and supplementary tables S1-S3) covers somatic endometrial-carcinoma recurrence, hotspot identity and in-silico outputs only; it contains no germline allelic-phase material and no PM3 rule, so it cannot supply the in-trans observation PM3 requires.
• No numeric threshold applies to PM3 in the supplied calibration data or in the governing framework; the criterion turns on an observation (variant in trans with a pathogenic variant) that no available source provides.

Still missing

• No proband-level genotype, zygosity or indication for testing is documented for c.6634A>G, so the allelic phase of the variant cannot be established.
• No second POLE variant (pathogenic, likely pathogenic or otherwise) is reported alongside c.6634A>G in any consulted source, so an in-trans partner can be neither identified nor excluded.
• No parental, sibling or other family genotypes are available from which the phase of c.6634A>G could be inferred.
• No homozygote and no somatic/tumour genotype co-occurring with this variant is reported (gnomAD homozygotes = 0; gnomAD v2.1 absent; the COSMIC search returned no record), so no indirect cis/trans signal exists either.
• The inheritance mode of the POLE-associated disorder being evaluated is not specified in this case (ClinVar condition 'not provided'; the gene-level artifact records no inheritance-mode entry), so the recessive setting PM3 presupposes is not documented.

Sources

Reasoning

PM6 (assumed de novo) requires that the variant be apparently de novo in an affected patient but without confirmation of parental biological relationship or without both parental genotypes - typically a single-parent test, absent non-paternity evidence, or a stated de novo observation without confirmatory trio testing. No governing gene-specific specification exists for POLE: no ClinGen CSPEC/VCEP rule set was found and the local custom framework (Leon-Castillo et al. 2020) customizes only PM1, PS4, PP3 and BP4, so PM6 falls back to generic ACMG/AMP 2015 (Richards et al.). This case contains no proband, no parental test result of any kind (neither confirmed nor assumed de novo), and no family data. The criterion therefore cannot be evaluated and is recorded as not_assessed with the absent family-level data identified as the gap.

Rule

Generic ACMG/AMP 2015 (Richards et al., PMID:25741868) PM6 - assumed de novo without confirmation of parental testing; no POLE gene-specific PM6 specification available.

Evidence considered

• Framework check: no ClinGen CSPEC/VCEP specification exists for POLE, and the local custom POLE framework (Leon-Castillo et al. 2020) customizes only PM1, PS4, PP3 and BP4; PM6 has no gene-specific specification, so generic ACMG/AMP 2015 (Richards et al., PMID:25741868) definitions apply.
• No proband, parental test result, or family history is present in case_summary.json, evidence.json, prefetch.json or the literature packet for this variant.
• ClinVar variation 1478557 lists a single 'not provided' condition with no family or de novo details.

Still missing

• No proband or affected individual is linked to this variant, so an apparently de novo observation cannot be established.
• No parental testing (even single-parent or unconfirmed) is reported.
• No family history is available to support the variant arising de novo.

Sources

Reasoning

PP1 (co-segregation with disease) requires evidence that the variant tracks with the disease phenotype across multiple affected family members, with strength scaled to the number of informative meioses and the strength of the segregation evidence. No governing gene-specific specification exists for POLE: no ClinGen CSPEC/VCEP rule set was found and the local custom framework (Leon-Castillo et al. 2020) customizes only PM1, PS4, PP3 and BP4, so PP1 falls back to generic ACMG/AMP 2015 (Richards et al.). This case contains no pedigree and no genotyped family members, so there are no meioses to score and no segregation pattern to evaluate. The criterion therefore cannot be evaluated and is recorded as not_assessed with the absent pedigree/meiosis data identified as the gap.

Rule

Generic ACMG/AMP 2015 (Richards et al., PMID:25741868) PP1 - co-segregation with disease in multiple affected family members; no POLE gene-specific PP1 specification available.

Evidence considered

• Framework check: no ClinGen CSPEC/VCEP specification exists for POLE, and the local custom POLE framework (Leon-Castillo et al. 2020) customizes only PM1, PS4, PP3 and BP4; PP1 has no gene-specific specification, so generic ACMG/AMP 2015 (Richards et al., PMID:25741868) definitions apply.
• No pedigree, no genotyped relatives, and no segregation data are recorded in case_summary.json, evidence.json, prefetch.json or the literature packet.
• ClinVar variation 1478557 provides only a single-submitter uncertain-significance call with no segregation information.

Still missing

• No pedigree is provided, so co-segregation of the variant with disease across a family cannot be assessed.
• No family member genotypes are available, so the number of informative meioses cannot be counted.
• Phenotype status of any relative is unknown, preventing segregation-with-disease evaluation.

Sources

Reasoning

PP4 (phenotype specificity) requires that the patient's phenotype or family history be highly specific for a disease with a single genetic aetiology. The León-Castillo 2020 custom POLE framework customizes only PM1, PS4, PP3 and BP4 and explicitly does not customize phenotype criteria, so generic ACMG/AMP applies. No proband phenotype description, HPO terms, indication for testing, age at onset, or family history was present in the case materials (case_summary.json, the clinical evidence bundle, and prefetch.json were checked; the only clinical-context field found was an empty ClinVar condition label 'not provided', and the single ClinVar submission is germline Labcorp testing with no condition recorded). Without the phenotype there is no basis on which to judge whether this individual's presentation is specific for POLE-related disease, so PP4 cannot be scored rather than scored negative. Recorded as not_assessed.

Rule

Generic ACMG/AMP 2015 PP4: patient's phenotype or family history is highly specific for a disease with a single genetic aetiology.

Evidence considered

• Generic ACMG/AMP PP4 definition applied because the León-Castillo 2020 custom POLE framework customizes only PM1/PS4/PP3/BP4 and explicitly excludes phenotype criteria.
• Case materials (case_summary.json, prefetch.json, clinical evidence bundle) contain no proband phenotype, HPO terms, indication for testing, or family history; the ClinVar submission (SCV002263622) lists no condition ('not provided').

Still missing

• No proband phenotype/family history (HPO terms, age at onset, indication for testing) is present in the case files, so phenotype specificity for POLE-related disease cannot be evaluated.
• POLE germline disease association itself is not established by an official framework in this case: the León-Castillo 2020 framework is somatic/endometrial-carcinoma derived and does not specify phenotype criteria; the PVS1 context file only weakly documents germline POLE loss-of-function disease literature.

Reasoning

Not assessed. BS3 under generic ACMG/AMP 2015 (the governing local POLE framework from Leon-Castillo et al. 2020 defines no PS3/BS3 rule) requires well-established functional studies showing no damaging effect on protein function. No functional assay of POLE p.Met2212Val - wild-type or variant - was identified in any retrieved source, so there is no experimental result that could support a benign functional claim. The benign-leaning signals present in this case are computational and therefore belong to BP4, not BS3: REVEL 0.212 (indeterminate, in the gray zone between the BP4 supporting <=0.29 and PP3 cut-offs) and BayesDel -0.035 (no verified published ACMG-strength calibration) are predictor outputs, not assays. OncoKB's 'Unknown Oncogenic Effect' with is_variant_specific = false is a record of absence, not a variant-specific determination of normal function, and its gene-level text concerns exonuclease-domain proofreading biology rather than this substitution. The single source triaged for this case (PMID:28492532) reports no functional data. Leon-Castillo et al. 2020 and its Supplementary Tables S1-S3 do not name p.Met2212Val; the paper's statement that a large majority of mutations outside the exonuclease domain are not pathogenic rests on somatic tumour genomic signatures (POLE-score, ultramutation, TMB) in endometrial carcinoma, not on an assay of this variant, and the framework itself specifies no BS3 rule. No ClinVar submission for this variant reports a non-damaging functional assay. Because no functional data exist in either direction, BS3 is not assessed rather than not met, and absence of functional evidence must not be converted into functional neutrality.

Rule

Generic ACMG/AMP 2015 (Richards et al., PMID:25741868): BS3 = well-established functional studies show no damaging effect on protein function. The governing local POLE framework (Leon-Castillo et al. 2020) specifies no BS3 rule and no official ClinGen POLE CSPEC/VCEP was available.

Evidence considered

• Governing framework: the local POLE framework (Leon-Castillo et al. 2020, J Pathol 250:323-335; custom_gene_framework, official_clingen = false) customizes PM1, PS4, PP3 and BP4 only, so BS3 is evaluated under generic ACMG/AMP 2015 (Richards et al., PMID:25741868), which requires well-established functional studies showing no damaging effect on protein function.
• No functional assay evidence for POLE c.6634A>G (p.Met2212Val) exists in any retrieved source. The only literature triaged for this case is PMID:28492532 (Sherloc), which reports no functional data and does not mention the variant; no full text was fetched (paper_extracts.json and fulltext_map.json are empty).
• OncoKB reports POLE M2212V as 'Unknown Oncogenic Effect' with is_variant_specific = false, not_reviewed = true and no citations - an explicit statement that no functional evidence exists, not evidence of normal function, so it cannot support BS3. Its gene-level background describes POLE exonuclease-domain ultramutation biology generally, not this substitution.
• Leon-Castillo et al. 2020 (PATH-250-323.pdf) was read directly and never mentions p.Met2212Val (0 matches for '2212'/'6634'); its only functional-type readout is the somatic POLE-ultramutated signature, which is restricted to exonuclease-domain variants, and the paper reports that only 1/23 (4%) of non-exonuclease-domain-mutant tumours showed it. These are tumour genomic observations, not a non-damaging assay of this variant, so they provide no BS3 evidence.
• Supplementary Tables S1-S3 (PATH-250-323-s002/s003/s004.xlsx) were opened directly and do not contain p.Met2212Val; they are somatic recurrence and in-silico prediction tables, not functional assays, and cannot support a benign functional claim.
• Computational predictors available for this variant: REVEL 0.212 (indeterminate; between the BP4 supporting <=0.29 and PP3 supporting >=0.644 cut-offs) and BayesDel -0.0354284 (no verified published ACMG-strength calibration available to this case). Predictor output is PP3/BP4 material and by definition cannot satisfy BS3; BayesDel is additionally not usable for any PP3/BP4 call here.
• No ClinVar submission for this variant reports BS3 or a non-damaging functional assay; the single submission (SCV002263622, Labcorp Genetics) classifies the variant Uncertain significance and cites only PMID:28492532, a methodology paper.
• No numeric threshold applies: BS3 is a qualitative evidence-existence criterion in ACMG/AMP 2015 and no POLE-specific BS3 calibration is provided by the governing materials.

Still missing

• No functional assay of POLE p.Met2212Val demonstrating preserved or normal protein function (proofreading fidelity, polymerase extension, mutator-complementation or equivalent) exists in the retrieved evidence, so the benign functional criterion cannot be satisfied.
• The governing local POLE framework (Leon-Castillo et al. 2020) defines no BS3 rule, is not an official ClinGen CSPEC, and no higher-priority POLE-specific BS3 specification was available.
• The only benign-leaning variant-level evidence is computational (REVEL 0.212; BayesDel -0.0354284); predictor output is PP3/BP4 material and must not be promoted to BS3.

Sources

Reasoning

BS4 (lack of segregation) requires evidence that the variant does not segregate with the disease phenotype in a family - specifically that affected family members lack the variant, arguing against pathogenicity. No governing gene-specific specification exists for POLE: no ClinGen CSPEC/VCEP rule set was found and the local custom framework (Leon-Castillo et al. 2020) customizes only PM1, PS4, PP3 and BP4, so BS4 falls back to generic ACMG/AMP 2015 (Richards et al.). This case contains no pedigree and no genotyped affected or unaffected family members, so non-segregation cannot be demonstrated or excluded. The criterion therefore cannot be evaluated and is recorded as not_assessed with the absent family-genotype data identified as the gap.

Rule

Generic ACMG/AMP 2015 (Richards et al., PMID:25741868) BS4 - lack of segregation with disease in a family; no POLE gene-specific BS4 specification available.

Evidence considered

• Framework check: no ClinGen CSPEC/VCEP specification exists for POLE, and the local custom POLE framework (Leon-Castillo et al. 2020) customizes only PM1, PS4, PP3 and BP4; BS4 has no gene-specific specification, so generic ACMG/AMP 2015 (Richards et al., PMID:25741868) definitions apply.
• No pedigree, no affected-relative genotypes, and no non-segregation data are present in case_summary.json, evidence.json, prefetch.json or the literature packet.
• ClinVar variation 1478557 provides only a single-submitter uncertain-significance call with no segregation or non-segregation information.

Still missing

• No pedigree is provided, so non-segregation of the variant with disease cannot be assessed.
• No genotypes from affected family members are available to test for absence of the variant.
• Phenotype status of any relative is unknown, preventing a lack-of-segregation evaluation.

Sources

Reasoning

BP2 is supporting benign evidence for a specific allelic configuration: the variant observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or observed in cis with a pathogenic variant under any inheritance pattern. It therefore requires a pathogenic partner allele whose phase relative to POLE c.6634A>G (p.Met2212Val) is known. No such observation exists in any source available for this variant. The governing local POLE package (Leon-Castillo et al. 2020) specifies only PM1, PS4, PP3 and BP4, and no ClinGen CSPEC/VCEP exists for POLE, so the generic ACMG/AMP 2015 BP2 definition applies. The ClinVar record (variation 1478557, VCV001478557) holds a single germline submission (SCV002263622, Labcorp Genetics; Uncertain significance; 1 star; no expert panel) with the condition recorded only as 'not provided', and states no co-occurring POLE variant, no zygosity and no phase. The trans limb presupposes a fully penetrant dominant disorder, and neither the case's ClinVar record (condition 'not provided') nor the gene-level information available for this case (an empty inheritance list) establishes full penetrance. The cis limb requires the variant to be phased on the same allele as a pathogenic variant, and no proband genotype, family genotype or phasing result exists in this case, which was reviewed at the variant level only. The population datasets contain no allele-configuration observation either: gnomAD reports zero homozygotes (v4.1 2/1,613,730 alleles, AF 1.24e-06; v2.1 absent), the gnomAD-Canada v1.0 query returned no record, and no somatic record of the variant was retrieved from COSMIC. Because neither limb can be tested from the available records, BP2 is recorded as not assessed rather than met or refuted.

Rule

Generic ACMG/AMP BP2 (PMID:25741868): observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or observed in cis with a pathogenic variant in any inheritance pattern; applied generically because the governing local POLE package specifies only PM1, PS4, PP3 and BP4 and no ClinGen CSPEC/VCEP exists for POLE.

Evidence considered

• Governing framework: the local POLE package (Leon-Castillo et al. 2020 custom POLE framework, official_clingen false) specifies only PM1, PS4, PP3 and BP4, and no ClinGen CSPEC/VCEP exists for POLE (cspec found = false), so BP2 was evaluated under the generic ACMG/AMP 2015 definition: observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder, or observed in cis with a pathogenic variant in any inheritance pattern (PMID:25741868).
• ClinVar variation 1478557 (VCV001478557) for NM_006231.4(POLE):c.6634A>G (p.Met2212Val): one germline submission (SCV002263622, Labcorp Genetics, formerly Invitae), Uncertain significance, 1 star, no expert panel, condition 'not provided'. The record describes a single variant and provides no co-occurring POLE variant, no zygosity, no phase and no family genotype, so neither the cis limb nor the trans limb of BP2 has an observation to rest on. The submitted classification was not used as criterion evidence.
• Penetrance and inheritance context: the trans limb of BP2 requires a fully penetrant dominant gene/disorder. Neither the case's ClinVar condition ('not provided') nor the gene-level artifact available for this case (empty inheritance list) establishes full penetrance, and the recognised biallelic POLE syndrome is recessive, so it cannot be confirmed that the trans limb could apply.
• Population and somatic data: gnomAD v4.1 2/1,613,730 alleles (AF 1.23936e-06; European non-Finnish 2/1,180,028, AF 1.69488e-06; homozygotes 0); gnomAD v2.1 returned no record; gnomAD-Canada v1.0 returned no record. No homozygote is reported, so the datasets contain no observation of this allele paired with a second POLE allele. The somatic (COSMIC) search returned no record of this variant from which any co-occurring alteration could be read.
• Case-level literature: the single PMID cited by the ClinVar submission (PMID:28492532, the Sherloc refinement of the ACMG/AMP criteria) was not available as full text for this case and no paper extract exists; it neither names this variant and its consequence nor provides co-occurrence, phase or segregation data that could bear on BP2.
• The local POLE package underlying the framework (PATH-250-323 PDF and supplementary tables S1-S3) addresses somatic endometrial-carcinoma recurrence, hotspot identity and in-silico output only and contains no BP2 rule and no germline phase material.
• No numeric threshold applies to BP2 in the supplied calibration data or in the governing framework; the criterion turns on a phased co-occurrence observation that no available source provides.

Still missing

• No phased genotype or pedigree data exist for c.6634A>G, so its cis/trans configuration relative to any potential pathogenic POLE variant is unknown.
• No co-occurring pathogenic or likely pathogenic POLE variant (germline or somatic) is reported with c.6634A>G in the ClinVar record or in any other consulted source.
• No homozygote or compound-heterozygous carrier of c.6634A>G is reported (gnomAD homozygotes = 0; v2.1 absent; the gnomAD-Canada query returned no record), so no allele-configuration observation exists; population datasets also carry no phenotype that could be attached to such an observation.
• The inheritance mode and penetrance of the POLE-associated disorder being evaluated are not specified for this case, so it cannot be determined whether the trans limb of BP2 (which requires a fully penetrant dominant disorder) could apply.

Sources

Reasoning

BP5 (variant found in a case with an alternate molecular basis for disease) is case-level evidence: it requires knowing whether the individual carrying this variant has another identified genetic or non-genetic cause that could explain their presentation. No such case-level information exists in the case materials - there is no proband phenotype, no indication for testing, no accompanying variant list or second molecular diagnosis, and no family data (case_summary.json, prefetch.json and the clinical evidence bundle were checked; the only ClinVar submission is a germline single-submitter VUS with no condition recorded). The variant is also absent from COSMIC and not reported as a somatic hotspot, so there is no tumour/case context invoking a second driver. With no case in which to look for an alternate molecular basis, BP5 cannot be evaluated and is recorded as not_assessed rather than negative. The León-Castillo 2020 framework does not customize BP5, so generic ACMG applies.

Rule

Generic ACMG/AMP 2015 BP5: variant found in a case with an alternate molecular basis for disease.

Evidence considered

• Generic ACMG/AMP BP5 definition applied because the León-Castillo 2020 custom POLE framework customizes only PM1/PS4/PP3/BP4.
• Case materials contain no case-level information (no phenotype, indication, family data, or list of co-occurring variants/second diagnosis) with which an alternate molecular basis could be identified; the single ClinVar submission records no condition.

Still missing

• No proband case data (phenotype, indication for testing, co-occurring variants or second diagnosis) are present in the case files, so BP5's requirement - a case with an alternate molecular basis for disease - cannot be tested.
• BP5 is not customized by the León-Castillo 2020 POLE framework; generic ACMG/AMP applies and requires case-level information that is absent.